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Supplementary Data from Targeting the mTOR Pathway for the Prevention of ER-Negative Breast Cancer

Supplementary Figure from Targeting the mTOR Pathway for the Prevention of ER-Negative Breast Cancer

Prevention of estrogen receptor (ER)-positive breast cancer is now possible using anti-estrogen drugs; however, this treatment is ineffective against ER-negative breast cancers. In this study, we hypothesized that inhibition of mTOR will suppress the growth of ER-negative and triple-negative breast cancers. To test the hypothesis, we used five ER-negative breast cancer models: MMTV-erbB2, C3 (1)/SV40TAg, p53-null mammary gland-transplant, p53-mutant mammary gland-transplant, and BRCA1co/co; MMTV-Cre+/+; p53+/– mouse models to determine whether the mTOR inhibitor everolimus is effective in preventing growth of ER-negative mammary tumors. Our study demonstrates that everolimus treatment significantly delays mammary tumor formation with varying degree in all five ER-negative mouse models. Everolimus treatment reduces the proliferation, with reduced phosphorylation of S6 kinase, and induces apoptosis of mammary tumor cells. In some of the p53-mutant mammary gland-transplant mice and C3 (1)/SV40Ag mice, everolimus completely prevents mammary tumor formation. Everolimus treatment also reduces proliferation of normal mammary gland cells. Our results support testing everolimus in clinical trials for the prevention of ER-negative breast cancer in women at high risk of ER-negative breast cancer. Prevention Relevance: Our results show that everolimus delays mammary tumor formation in multiple mouse models, suggesting that mTOR inhibitors will be useful for the prevention of ER-negative and triple-negative breast cancer in humans. See related Spotlight, p. 787

Background: Recent research in breast biology supports the cancer stem cell hypothesis which asserts that malignancies arise in tissue stem cells through dysregulation of self-renewal. The cancer stem cell hypothesis has important implications for early detection, prevention and treatment of breast cancer. If breast cancers arise from the transformation of normal stem cells, it may be possible to target stem cells for the prevention of cancer. We have previously shown that rexinoids prevent the development of ER-negative breast cancer in transgenic mouse models. Previous studies have demonstrated a role of retinoid signaling in the regulation of normal breast stem cell self-renewal and differentiation. Bexarotene and LG100268 are rexinoids that selectively bind RXR receptors. Based on the effects of these molecularly targeted agents, we hypothesized that rexinoids prevent breast cancer development by suppressing the growth and transformation of the breast stem cell population. Methods: We performed in vivo and in vitro studies to address our hypothesis. Cell proliferation assays were used to determine whether rexinoids can inhibit the growth of normal, pre-malignant and malignant breast cell lines (HMECs, MCF10A, DCIS.COM, SUM225, HCC1937 and HCC38 cell lines). To determine whether these agents affect stem cells, we performed mammosphere assays and FACS analysis after treatment with vehicle or rexiniods. We next conducted in vivo studies using MMTV-Wnt transgenic mice treated with LG100268 (50mg/kg) or vehicle, and studied the stem cell properties of mammary epithelial cells from these mice. The stem cell population in mammary glands was quantified by staining of CD24, CD49f and using FACS analysis and mammosphere assays. We also performed limiting dilution assay to demonstrate the in vivo effect of LG100268 on stem cell population. Fvb mice injected with 1,10,100,1000 wnt tumor cells were treated with vehicle or LG100268 and tumor outgrowth rate is recorded in each group. Results: Our results showed that the rexionid LG100268 inhibited the growth of breast cell lines only at high concentrations (IC50>10uM) in most of the tested cell lines. Results of in vitro mammosphere assays showed that a dose of LG100268 (100nM) much lower than IC50 reduced mammosphere formation efficiency by more than 40% in all breast cancer cell lines. Results from our in vivo experiments showed that LG100268 reduced the mammary epithelial stem cell population by 57% as defined by the CD24+/CD49f++ population identified by FACS and by 47% as measured by in vitro mammosphere assays. In addition in vivo limiting dilution assay studies demonstrate slower tumor outgrowth rate and lower repopulation frequency in groups with LG100268 treatment. Conclusion: These results from in vitro and in vivo studies demonstrate that the rexinoid LG100268 is a potent mammary stem cell inhibitor. This study shows that rexinoids suppress stem cell expansion and suggest that mammary stem cell inhibitors will be useful breast cancer preventive agents. This work is supported by a grant from the Breast Cancer Research Foundation (to PHB) and from an NIH/NCI RO1 grant (CA078480) (to PHB). This abstract is also presented as Poster B30. Citation Format: Jing Zhao, Yun Zhang, Jamal L. Hill, Abhijit Mazumdar, Ivan Uray, Reid P. Bissonnette, Powel H. Brown. Targeting breast cancer stem cells using cancer preventive rexinoids. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr PR-06.